Brief Note: Effects of Prostaglandins, Epinephrine and NaF on Human Leukocyte, Platelet and Liver Adenyl Cyclase

Despite the limited scope of this study, the data show that homogenates ofhuman leukocytes, platelets and liver are capable of synthesizing cyclic AMP,and that such preparations are responsive to hormonal stimulation. Epinephrine stimulates cyclic AMP production in liver and leukocyte homogenates,and NaF shows a stimulatory effect in each tissue.^1,8 The data confirm theprevious reports^10,11 that prostaglandin E₁ stimulates platelet adenyl cyclase,but further shows a lack of stimulation by prostaglandin F/₁. Incubation ofleukocytes with the prostaglandins showed a similar effect.

It is suggested that further study may establish a relationship betweenadenyl cyclase stimulation and chemotaxis or phagocytic activity of circulatingleukocytes as has been reported in amebae.¹² Since systemic enzyme deficienciessuch as phosphorylase in Hers’ Disease¹³ and amylo-1.41.6-transglucosidasein Andersens’ Disease¹⁴ are evident in leukocytes, it is suggested that furtherstudy of circulating leukocytes may also detect an abnormal responsiveness ora deficiency of adenyl cyclase.

Submitted on August 8, 1969 Accepted on December 29, 1969     

Leukapheresis in Man. III. Hematologic Observations in Patients with Leukemia and Myeloid Metaplasia

Eleven patients with leukemia and one patient with myeloid metaplasiaunderwent leukapheresis on 18 occasions for 94 to 275 minutes during which93 to 1668 x 10⁹ leukocytes were removed. All patients exhibited a significantand continued decline of peripheral leukocyte concentration during or afterthe procedure. In 12 of the 18 instances, the leukocyte concentration returnedslowly to the initial leukocyte level within 1 hour to 22 days. The numberof leukocytes withdrawn represented 16 to 247 per cent of the initial circulatingvolume removed at a rate of 0.13 to 1.14 leukocyte blood volumes per hour.The RAR was 1:1 to 12:1 to the circulating leukocyte number. Rate of replenishment of the circulating immature leukocyte numbers were 4.0 x 10⁷to 52.2 x 10⁹/Kg./day. The PMN’s were replaced at rates of 10 x 10⁶ to 7.05x 10⁹/Kg./day which were equal to or slower than in normal subjects. Changesin number occurred in the dominant leukemic cell types without significantshifts in the differential counts. No changes in marrow population other thana slight decrease in cellularity were observed.

The data indicate that in the leukemic patient the peripheral leukocyte concentration was not maintained or replenished promptly following the withdrawal of sizeable quantities of leukocytes, demonstrating a block in transferof leukocytes from the tissues to the blood. This is in marked contrast to theleukocytosis and marrow stimulation observed in hematopoietically normalsubjects following leukaphereses.

The platelet counts fell promptly during leukapheresis, returning towardcontrol levels in eight studies within 7 hours following the procedure. In fourstudies the platelet counts returned to control levels in 3 to 9 days. Thechanges in platelet concentrations were similar to those observed withhematologically normal subjects. The size of the platelet reservoir in theseleukemic patients is about twice that of the circulating blood.

Submitted on May 14, 1962 Accepted on October 14, 1962     

Studies of Leukocyte Alkaline Phosphatase in Mongolism: A Possible Chromosome Marker

Leukocyte alkaline phosphatase was measured by a biochemical method ina group of 58 mongolian idiots. The mean activity found in the 41 childrenless than 10 years of age was equivalent to 89 mg. of phosphorus per 10¹⁰neutrophilic leukocytes per hour. This was compared to the value of 66 mg.of phosphorus per 10¹⁰ neutrophilic leukocytes per hour obtained in a groupof 41 control children in the same age group (after correction of the controlmean to the same mean age). The difference is significant at the 0.5 per centlevel of confidence.

This difference was interpreted as confirming (but not proving) the hypothesis that leukocyte alkaline phosphatase formation is controlled by a geneon chromosome number 21—the chromosome for which mongolian idiots aretrisomic. The hypothesis arose because of the known deficiency of this enzymein the leukocytes of patients with chronic granulocytic leukemia, and theknown partial deletion of chromosome 21 in this disease.

This finding should provide a stimulus to further investigations into thecontent of alkaline phosphatase in leukocytes, possible polymorphism of leukocyte alkaline phosphatases, linkages with other inherited traits, and relationships between leukocyte and other tissue phosphatases.

Submitted on December 10, 1962 Accepted on February 10, 1963     

SINGLE CELL IMAGING REVEALS ABNORMAL INTRACELLULAR CALCIUM SIGNALS WITHIN RHEUMATOID SYNOVIAL NEUTROPHILS

Intracellular calcium (Ca²⁺) signalling in synovial fluid (SF)polymorphonuclear leucocytes(PMN) from patients with rheumatoidarthritis (RA) was compared to RA and normal circulating bloodPMN using single cell imaging. RA SF PMN stimulated by the peptidef-Met-Leu-Phe (FMLP) showed a striking difference in the releaseof Ca²⁺ from the intracellular store compared to RA and normalcirculating blood PMN. Stimulation caused the release of a verydispersed, nonrestricted ‘cloud’ of Ca²⁺ in 60%of RA SF PMN compared to the highly localized and restricted‘cloud’ observed in only 30% of normal circulatingPMN. In the presence of extracellular Ca²⁺, both RA SF and normalblood PMN showed heterogeneity in both the timing and magnitudeof their cytosolic free Ca²⁺ signalling. These observationsimply that the Ca²⁺ signalling mechanism in RA SF and RA bloodPMN has been primed in a way which could exacerbate the releaseof inflammatory mediators. This may have serious implicationsfor explaining the aberrant behaviour of SF PMN in RA. KEY WORDS: Rheumatoid arthritis, Polymorphonuclear leucocytes, Synovial fluid, FMLP, Cytosolic free Ca²⁺, Intracellular Ca²⁺ ‘cloud’     

Human Leukocyte Metabolism in Vitro: II. The Effect of 6-Mercaptopurine on Formate-C14 Incorporation into the Nucleic Acids of Acute Leukemic Leukocytes

1. The effects of added 6-mercaptopurine (6-MP) on the in vitro incorporation of radioformate (C¹⁴) into the leukocyte nucleic acid purines andthymine of six cases of acute myeloblastic leukemia (AML) and four cases ofacute lymphoblastic leukemia (ALL) have been compared with the patients’clinical response to 6-MP.

2. In seven cases subsequent therapy with 6-MP produced leukopenia. Ofthese, in vitro 6-MP diminished leukocyte C¹⁴ incorporation into the nucleicacid purines in three cases of AML, had no effect in two cases of AML andone case of ALL, and enhanced C¹⁴ incorporation into the leukocyte purines ofa second case of ALL. Thymine synthesis was slightly diminished by 6-MP inthe AML leukocytes in which purine synthesis was inhibited, and in one drugsensitive ALL leukocyte population in which purine synthesis was slightlyincreased by the analog. Thus, no regular and consistent relationship betweenthe antileukemic effect of the drug and suppression of purine or thyminesynthesis by 6-MP in vitro could be demonstrated.

3. Studies of the leukocytes of two cases of drug-resistant ALL yieldedresults similar to those observed in presumably drug-sensitive ALL cells.

4. The leukocytes of two cases of AML with acquired drug-resistanceshowed an increased capacity for in vitro RNA purine synthesis; in the onecase in which studies before and after the development of resistance werepossible, this property was apparently acquired during therapy. This suggeststhat an increase in purine biosynthetic enzyme reserves may be a mechanismof 6-MP-resistance in some human leukemias.

Submitted on January 24, 1966 Accepted on April 4, 1966     

Protein Synthesis in Rat Lymphocytes: Radioautographic Studies of Availability and Utilization of Labeled Amino Acids in Vivo

Injections of H³-methionine and H³-leucine were combined with radiochemical and radioautographic technics to study the availability time ofH³-methionine and the protein synthetic ability of rat lymphocytes in vivo.

Although 98.5 per cent of H³-methionine was removed from the serum5 minutes after injection, sufficient quantities persisted and/or re-entered theserum from tissues to cause increasing grain counts in radioautographs oflarge lymphocytes for 1 hour after isotope administration. A small amount ofadditional labeling occurred during the 2nd hour, but it is calculated thatlabeling is 97-98 per cent complete by 1 hour.

All of the large and medium lymphocytes were labeled in the thymus, lymphnode, and thoracic duct lymph at short intervals after injection of 4 µc./Gm.body weight of H³-methionine. Evidence is presented that protein synthesisoccurs in the nucleus as well as in the cytoplasm and that newly formed protein is equally distributed between daughter cells following mitosis. Previousimmunochemical studies are combined with information on generation timeand disappearance rates of radioactivity to suggest that large and mediumlymphocytes are constantly producing and releasing proteins. Large andmedium cells in lymph and lymph node are more active in this than aresimilar cells in the thymus. Evidence of reutilization of labeled metabolitesin the lymph node and especially in the thymus is discussed.

Although not all small lymphocytes were labeled by 4 µc./Gm. body weightof H³-methionine, it was shown that larger doses of isotope would label 100per cent of them. Small lymphocytes in thoracic duct lymph evidenced significant turnover of labeled protein during the 1st day after isotope administration.

Submitted on August 21, 1963 Accepted on November 9, 1963     

The Detection and Quantification of Rh(D) Antigen Sites on Human Leukocytes

Radiolabeled eluates of human anti-D were used to measure the capacityof leukocytes to bind the D antibody in cell suspensions prepared from 16normal and 3 leukemic bloods from Rh(D) donors. The contamination of theleukocyte suspensions by D positive red cells was measured and the contribution of D antigen sites by these cells was estimated. After correction wasmade for the D antibody bound by the contaminant red cells, no specific binding of D antibody by Rh(D) leukocytes could be detected. Three pairs ofRh(D) and rh(d) leukocyte dilution curves of I¹³¹-labeled anti-D uptakewere compared with the uptake by D positive and D negative red cell dilutions. No significant differences among the D negative erythrocyte, the rh(d)leukocyte and the Rh(D) leukocyte curves were obtained. The results werecollated with previous serologic evidence concerning the presence of ABOand Rh antigens on human leukocytes.

Submitted on April 16, 1963 Accepted on June 7, 1963     

The Antigenicity of Normal and Leukemic Human Leukocytes

1. A leukoagglutinin was formed in the serum of a normal human subjectwho received 10 intravenous injections of blood from a single patient withchronic myelogenous leukemia over a period of 20 weeks.

2. Coincident with development of the leukoagglutinin, first detectable oneweek after the fifth injection of leukemic blood, the normal subject experiencedprogressively more severe febrile reactions to the infusions and exhibited acharacteristic pattern of leukocyte response—namely, an immediate transientleukopenia, followed by a leukocytosis which reached its peak around 3 hoursand subsided to normal within 12 hours.

3. During the early part of the investigation immature leukocytes, presumably from the leukemic donor, could be identified in the recipient’s circulationduring the first hour immediately following injection, but none could befound following the tenth infusion of leukemic blood.

4. The leukoagglutinin which appeared in response to the injections of bloodfrom the single leukemic donor was a typical iso-antibody, showing a broadpattern of reactivity against normal leukocytes from 127 of 129 donors, leukemicleukocytes from 5 of 5 patients with chronic myelocytic leukemia and 6 of 6patients with chronic lymphocytic leukemia. No reactivity was observed againstthe recipient’s own leukocytes, and little or no reactivity was demonstrableagainst the immature leukocytes from 3 patients with acute leukemia.

5. Eighteen months after the last injection of leukemic blood, restimulationof a leukocyte iso-agglutinin in the previously immunized recipient was provoked within one week of commencing a series of intravenous infusions ofblood from a single normal donor.

6. The volume of normal leukocytes employed for the restimulation was 1/10to 1/100 the volume of leukemic leukocytes employed for the primary immunization.

7. The concept of antibody excess was demonstrable in the sensitized recipient. No evidence of in vivo absorption of leukoagglutinin activity was observedafter transfusion of 500 ml. of blood from the normal donor. The severity ofthe recipient’s reaction to the transfused blood was clearly related to the doseof donor leukocytes administered, 0.47 billion cells causing no reaction but4.16 billion causing a moderately severe reaction.

8. Fifteen months after completion of the injections of normal blood, reexposure of the normal subject to injections of blood from a second leukemicdonor resulted in prompt restimulation of leukoagglutinin activity in therecipient’s serum.

9. The leukoagglutinin could be completely absorbed in vitro by incubationwith donor leukocytes.

10. The leukoagglutinin was concentrated in the gamma globulin fraction ofthe recipient’s plasma.

11. The recipient exhibited typical symptomatic reactions and transient hematologic changes following the infusions of leukemic blood.

12. It was possible to correlate the severity of the recipient’s clinical reactionsboth with the strength of the recipient’s leukoagglutinin, as well as with thedose of donor leukocytes transfused.

13. Serologic observations, plus the results of fractionated transfusion studies,indicated that the recipient’s transfusion reactions were related to sensitivityto the donor’s buffy coat (Part II), and more specificially to donor leukocytes(Part III), rather than to donor plasma, platelets or erythrocytes.

14. Sustained stimulation of the recipient’s white cell count as a result of theinjections of leukemic blood was not observed.

15. There has thus far been no evidence of transmission of leukemia to therecipient (now 6 years after the first course of injections of leukemic bloodand 2 years since completion of the present study).

Submitted on July 15, 1960 Accepted on November 20, 1960     

Immunologic and Cytogenetic Studies of Chronic Lymphocytic Leukemic Cells

The lymphocyte transformation response of 17 chronic lymphocytic leukemiapatients when tested in the short-term tissue culture with PHA-M, and PPDwas found to be significantly decreased when compared to normal subjects.Serum factors were not found to be responsible for this cellular hyporesponsiveness. The proportions of immunoresponsive lymphocytes found in thepatients’ peripheral circulation decreased as their white blood cell count increased. The transformation response to PHA-M was generally better thanto PPD. Neither the PPD negative patients nor the normal PPD negativesubjects’ cells responded to PPD stimulation in vitro.

Monocytes usually would phagocytize particles added to the cultures andcould thus be distinguished from the nonphagocytic proliferating lymphocyteswhich were the only cells that incorporated thymidine H³. Radioautographsof tritiated thymidine also revealed the rate of PPD lymphocyte transformationto be slower than with PHA-M. There were no significant differences in theproportions or the degree of leukemic and normal transformed lymphocytelabeling with tritiated thymidine.

Cytogenetic studies revealed that the patients’ mitotic indices both in vivoand in vitro were markedly depressed. The modal chromosome number was46 in each patient, and no cytogenetic abnormalities other than those due toexposure to radiation were found.

Submitted on August 26, 1964 Accepted on November 22, 1964     

The Duffy Blood Group System in Transfusion Reactions: A Review of the Literature and Report of Four Cases

1. The literature on the incidence and inheritance of the "Duffy" bloodgroup factors and their significance in blood transfusion and erythroblastosisfetalis is briefly reviewed.

2. Four new cases in which anti-Fy^a was detected are reported.

3. The necessity for the use of the Coombs’ compatibility test with apotent Coombs’ serum to prevent reactions due to anti-Fy^a is re-emphasized.

4. The inadequacy of a 15 minute incubation period for the Coombs’ compatibility test in certain cases is noted.

5. The desirability of specific identification of an antibody detected ina patient’s serum prior to transfusion whenever possible is suggested.

Submitted on January 31, 1959 Accepted on March 18, 1959     

Studies of Abnormal Leukocyte Bodies in the Mink

The occurrence of abnormal bodies in the cytoplasm of neutrophils, eosinophils, monocytes and lymphocytes of Aleutian (aa) mink is reported. Similarities of these bodies to the leukocyte abnormalities of Chediák-Higashi syndrome of children are pointed out.

Submitted on January 3, 1963 Accepted on April 23, 1963     

Lymphocyte and Granulocyte Enzyme Activity in Patients with Down's Syndrome

Patients with trisomic Down’s syndrome were found to have significantincreases of acid phosphatase, alkaline phosphatase, and glucose-6-phosphatedehydrogenase in both lymphocytes and polymorphonuclear leukocytes separated from white blood cells by the procedure of Rabinowitz. The alterationin enzyme activities appears not to be directly related to genes located on thechromosome causing Down’s syndrome.

Submitted on March 2, 1967 Accepted on April 13, 1967     

The Recovery of Lethally Irradiated Dogs Given Infusions of Autologous Leukocytes Preserved at -80 C

Nine normal mongrel dogs were exposed to 1200 r whole-body irradiationat 4 to 5 r per minute. They were then given intravenous infusions of 2.12 to20 x 10⁹ autologous leukocytes that had been previously stored at -80 C. in10 per cent dimethyl-sulfoxide.

Three dogs survived with delayed but complete hematopoietic recovery.Three showed beginning marrow regeneration but died within 3-4 weeks ofirradiation. Three given less than 6 x 10⁹ cells died within 21 days. The numberof leukocytes infused was critical since there was no survivor among the dogsreceiving less than 9 x 10⁹ cells.

It is concluded that peripheral blood contains primitive cells capable ofrepopulating marrow spaces and restoring marrow function.

Submitted on May 8, 1963 Accepted on June 27, 1963     

Malic and Lactic Dehydrogenase Isozymes of Normal and Leukemic Leukocytes Separated on Glass Bead Columns

Five LDH and two MDH isozyme bands were obtained with acrylamidegel electrophoresis of leukocyte extracts. Normal lymphocytes showed a hightotal H-LDH (heart type) activity (67 per cent) with 25 per cent in LDH-1and only 3.5 per cent in LDH-5. Lymphocytes from chronic lymphatic leukemia(CLL) and lymphosarcoma leukemia (LSA-LL) had less LDH-1, and moreLDH-3 and LDH-4, than normal lymphocytes. The H-LDH fell to 60.5 per centin CLL and 56 per cent in LSA-LL. PMN leukocytes had low H-LDH activity(38.8 per cent) with 3.3 per cent in LDH-1 and 25.8 per cent in LDH-5. Inmyelogenous leukemia, myeloblasts had the most LDH-1 and H-LDH, whilemature PMN had the least. PMN leukocytes isolated from CLL, LSA-LL, andmyelogenous leukemia had LDH patterns like the normal. Monocytes fromacute monocytic leukemia were low in LDH-1 and LDH-5, but had a high totalenzyme content. They evidently were rich in LDH-2, 3, and 4.

Lymphocytes had less MDH-1 (60 per cent) than PMN leukocytes (78 percent). In CLL, lymphocyte MDH-2 increased. In myelogenous leukemia,myeloblasts had the most MDH-2 and mature PMN the least. Monocytesfrom monocytic leukemia contained a little more MDH-2 than PMN leukocytes.

In general, white cell immaturity and/or ability to divide was associatedwith high levels of LDH-1, total H-LDH, and MDH-2.

Submitted on May 5, 1966 Accepted on June 26, 1966     

Specificity of lytic factors for erythrocytes,leukocytes and platelets in a case of pancytopenia

A case of chronic idiopathic pancytopenia in a young girl is presented, in whichthe pancytopenia was shown to be due to increased destructions of all 3 blood celltypes. Antileukocyte and antiplatelet antibodies were demonstrated by transfusion methods as well as by in vitro agglutination, while differential agglutination provided evidence of a plasma factor causing increased red cell destruction.

Cross absorption experiments demonstrated the presence in the patient’sserum of at least 2 separate and distinct antibodies, specific for leukocytes andplatelets respectively.

Observations on the phagocytic behavior of leukocytes and on the electrophoretic mobility of leukocytes and platelets exposed to the patient’s serum arereported.

Submitted on October 21, 1955 Accepted on March 24, 1956     

Relatively Selective Beta Irradiation of Lymphatic Structures in the Dog Using Y90-DTPA

In an attempt to provide relatively selective irradiation of tissues responsible for the homograft rejection response and to minimize exposure of other radiosensitive tissues, we developed a technic using internally administered Yttrium-90 chelated with DTPA (diethylene triamine-pentaacetic acid). Dogs given sublethal doses of Y⁹⁰-DTPA radiation had a benign clinical course, the only remarkable finding being a selective lymphopenia without depression of granulocytes, platelets, or reticulocytes. Dogs lethally irradiated with Y⁹⁰-DTPA showed depression of all formed blood elements, and severe depression of lymphocytes was most prominent. As the homograft rejection response appears to be a function of lymphatic tissues, the results obtained suggest the use of this procedure to prepare large mammals for homologous tissue transplantation.

Submitted on March 23, 1963 Accepted on September 6, 1963     

Effects of platelet-activating factor on cardiovascular function,oxygen free radical status,and blood chemistry

Platelet-activating factor (PAF) is released in numerous clinical situations. PAF primes or directly activates polymorphonuclear (PMN) leukocytes, which results in release of oxyradicals (O ₂ ^– , H₂O₂, .OH) and hypochlorous acid (HOCl). The authors investigated the effects of PAF (1 µg/kg IV) in the absence and in the presence of antioxidants (superoxide dismutase [SOD], catalase [CAT], dimethylthiourea [DMTU]) and methionine, a quencher of HOCl, on cardiac function and contractility; blood lactate, gases, and pH levels; serum creatine kinase activity (CK); chemiluminescent activity of PMN leukocytes; and cardiac tissue malondialdehyde (MDA) in anesthetized dogs. Hemodynamic measurements and collection of blood samples for various biochemical measurements were made before and at various intervals up to two hours after PAF administration in the presence and absence of various antioxidants.PAF produced a decrease in indices of cardiac function and contractility and an increase in systemic and pulmonary vascular resistance. There were decreases in the blood pH and PMN leukocyte chemiluminescence and increases in blood lactate, serum CK activity, and tissue MDA content. SOD plus catalase or DMTU plus methionine reduced the effects of PAF on cardiac function and contractility, blood lactate and pH, serum CK, and cardiac tissue MDA. The antioxidants only partially antagonized the deleterious effects of PAF. The combination of SOD + CAT was superior to that of DMTU + methionine in reducing the deleterious effects of PAF.These results suggest that PAF-induced depression of cardiac function and contractility, and the increase in systemic and pulmonary vascular resistance, may be partly mediated by the release of oxyradicals and HOCl from PMN leukocytes. Antioxidants may be beneficial in reducing the deleterious effects of PAF on the cardiovascular system.     

The Carbohydrate Metabolism and Respiration of Isolated Small Lymphocytes: In Vitro Studies of Normal and Phytohemagglutinin Stimulated Cells

1. A homogeneous population of small lymphocytes with an average size of6.7 micra was isolated from equine blood.

2. These cells could be maintained in vitro, with essentially complete survivalfor 24 hours, and with a 50% viability for five days.

3. The small lymphocytes consumed glucose at a rate of 1.87 mµmoles/1 x10⁷ cells/minute, and produced lactic acid at a rate of 2.30 mµmoles/1 x 10⁷cells/minute.

4. The oxygen consumption of small equine lymphocytes was 1.023 ± .165mµmo1es oxygen 1 x 10⁷ cells/minute, and that of mixed peripheral bloodleukocytes, 1.25 ± .07 mµmoles oxygen/1 x 10⁷ cells/minute, as determined, using a Clark oxygen electrode.

5. Lymphocyte glycolysis was stimulated under anaerobic conditions (Pasteur effect), and viability appeared unimpaired after 24 hours in a N₂ environment.

6. Uncoupling of oxidative phosphorylation by the addition of 2,4, dinitrophenol stimulated both respiration and glycolysis.

7. The glycogen content of the normal small horse lymphocyte was 7.45 ±1.04 µg glucose equivalents per 1 x 10⁷ cells.

8. Many of the initial cell population were transformed into "blasts" followingthe addition of phytohemagglutin to the tissue culture medium. Thisresponse was associated with an increase in the rate of glycolysis andrespiration by 24 hours, and a rise in intracellular glycogen by 48 hours.

Submitted on December 7, 1966 Accepted on May 22, 1967     

Studies in Bisalbuminemia: Binding Properties of the Two Albumins

1. An abnormal serum protein pattern in a patient with Wegener’s granulomatosis and five of his relatives was identified as bisalbuminemia by electrophoretic and immunochemical methods.

2. With the exception of the patient with Wegener’s syndrome, the presenceof bisalbuminemia was not associated with a significant change in total serumproteins, total albumin, serum components other than albumin, or any disease.

3. Addition of I¹³¹-thyroxine to bisalbumin sera resulted in thyroxine binding by albumin B but not by albumin A. The failure of albumin A to bindadded I¹³¹-thyroxine leads to speculation that, in this family, neither albuminA nor B are identical to normal human serum albumin.

Submitted on December 4, 1961 Accepted on April 17, 1962     

Leukokinetic Studies. VII. Morphology of the Bone Marrow and Blood of Dogs Given Vinblastine Sulfate

The effect of a single injection of vinblastine sulfate was studied in 50mongrel dogs. Nine of 34 dogs given 0.2 mg./Kg. of VLB died with gastrointestinal toxicity and the mortality rate increased as the dosage of VLB wasincreased. The morphologic pattern of leukocyte suppression and recovery inthe bone marrow and blood was studied in detail in surviving animals.

The cells of the bone marrow were markedly affected by VLB. Within 4hours there was an increase in the number of cells in metaphase and, by day1, virtually all proliferating leukocytes and erythrocytes had disappeared. Anorderly repopulation of the bone marrow followed.

The neutrophils, eosinophils, lymphocytes and monocytes of the blood wereall markedly altered in concentration after VLB. Each type of cell first decreased to abnormally small numbers and then increased to abnormally largenumbers in the blood. The curve of disappearance from and reappearance inthe blood differed for each cell type.

The changes in blood neutrophil number and morphology were correlatedwith changes in the blood neutrophil precursor cells of the marrow. The following conclusions were reached concerning the neutrophils and the assumptions implicit to these conclusions were detailed.

1. In the dog, the marrow contains enough post-mitotic granulocytes toreplace those lost from the blood for at least 3 to 4 days.

2. The release of mature neutrophils from the bone marrow is a functionof the rate at which blood neutrophils are lost and proceeds normally evenwhen the marrow granulocyte reserve is partially depleted.

Submitted on March 27, 1963 Accepted on August 20, 1963